The present invention relates to the field of bioconjugate preparation, and more particularly, to a class of bifunctional boronic compound complexing reagents useful for the conjugation of biological macromolecules, and the method of making and using such reagents.
Bioconjugation is a descriptive term for the joining of two or more different molecular species by chemical or biological means, in which at least one of the molecular species is a biological macromolecule. This includes, but is not limited to, conjugation of proteins, peptides, polysaccharides, hormones, nucleic acids, liposomes and cells, with each other or with any other molecular species that add useful properties, including, but not limited to, drugs, radionuclides, toxins, haptens, inhibitors, chromophores, fluorophores, ligands, etc. Immobilization of biological macromolecules is also considered a special case of bioconjugation in which the macromolecule is conjugated, either reversibly or irreversibly, to an insoluble solid phase support. Bioconjugation is utilized extensively in biochemical, immunochemical and molecuar biological research. Major applications of bioconjugation include; detection of gene probes, enzyme-linked immuno solid-phase assay, monoclonal antibody drug targeting and medical imaging.
Bioconjugates are generally classified as either direct or indirect conjugates. Direct conjugates encompass those in which two or more components are joined by direct covalent chemical linkages. Alternatively, indirect conjugates encompass those in which two or more components are joined via an intermediary complex involving a biological macromolecule. The system described herein is the first to enable the formation of indirect conjugates without dependence upon an intermediary biological macromolecule.
Although numerous methods of indirect bioconjugate preparation have been described, a significant number of those reported in the literature have been prepared by exploiting the Avidin-Biotin system, in which, the binding specificity of the protein Avidin (purified from egg white), or Streptavidin (purified from the bacterium Streptomyces avidinii), toward the cofactor Biotin (vitamin H) is utilized to bridge an Avidin conjugated macromolecule with a biotinylated macromolecule. Both Avidin and Streptavidin possess four Biotin binding sites of very high affinity (Ka=1015 Mxe2x88x921).
The Avidin-Biotin system has been utilized extensively for enzyme-linked immuno solid-phase assay (ELISA), in which an enzyme-Avidin conjugate (useful for detection by reaction with the enzyme""s substrate to afford a colored or chemiluminescent product) is employed to detect the presence of a biotinylated antibody, after first binding the antibody to an immobilized antigen or hapten. Applications of the Avidin-Biotin system number in the hundreds, and have recently been reviewed (Wilchek, M. and Bayer, E. A., (1990) Methods in Enzymology, 184).
Although utilized extensively, several limitations are known to be associated with the Avidin-Biotin system, which include nonspecific binding generally attributed to the basicity of the Avidin molecule, nonspecific binding attributed to the presence of carbohydrate residues on the Avidin molecule, and background interference associated with the presence of endogenous Biotin, which is ubiquitous in both eukaryotic and prokaryotic cells.
An alternative indirect bioconjugation system designed to overcome some of the limitations associated with the Avidin-Biotin system has recently been developed for the detection of gene probes by ELISA (Kessler, C., Hxc3x4ltke, H.-J., Seibl, R., Burg, J. and Mxc3xchlegger, K., (1990) Biol. Chem. Hoppe-Seyler, 371, 917-965). This system involves the use of the steroid hapten Digoxigenin, an alkaloid occuring exclusively in Digitalis plants, and Fab fragments derived from polyclonal sheep antibodies against Digoxigenin (anti-Digoxigenin). The high specificity of the various anti-Digoxigenin antibodies affords low backgrounds and eliminates the non-specific binding observed in Avidin-Biotin systems. Digoxigenin-labeled DNA and RNA probes can detect single-copy sequences in human genomic Southern blots. The development of the Digoxigenin anti-Digoxigenin system has recently been reviewed (Kessler, C. (1990) in Advances in Mutagenesis Research (Obe, G. ed.) pp. 105-152, Springer-Verlag, Berlin/Heidelberg). The Digoxigenin anti-Digoxigenin system is the most recent representative of several hapten-antibody systems now utilized extensively for bioconjugation.
Phenylboronic acids are known to interact with a wide range of polar molecules having certain requisite functionalities. Complexes of varying stability, involving 1,2-diols, 1,3-diols, 1,2-hydroxy acids, 1,3-hydroxy acids, 1,2-hydroxylamines, 1,3-hydroxylamines, 1,2-diketones and 1,3-diketones, are known to form with either neutral phenylboronic acid or phenylboronate anion. Consequently, immobilized phenylboronic acids have been exploited as chromatographic supports to selectively retain, from diverse biological samples, those molecular species having the requisite functionalities. Many important biological molecules including carbohydrates, catecholamines, prostaglandins, ribonucleosides, and steroids contain the requisite functionalities, and have been either analyzed or purified in this manner. The use of phenylboronic acid chromatographic media for the isolation and separation of biological molecules has been discussed in several reviews (Singhal, R. P. and DeSilva, S. S. M. (1992) Adv. Chromatog., 31, 293-335; Mazzeo, J. R. and Krull, I. S. (1989) BioChromatog., 4, 124-130; and Bergold, A. and Scouten, W. H. (1983) in Solid Phase Biochemistry (Scouten, W. H. ed.) pp. 149-187, John Wiley and Sons, New York).
Phenylboronic acid, like boric acid, is a Lewis acid, and ionizes not by direct deprotonation, but by hydration to give the tetrahedral phenylboronate anion (pKa=8.86). Phenylboronic acid is three times as strong an acid as boric acid. Ionization of phenylboronic acid is an important factor in complex formation, in that, upon ionization, boron changes from trigonal coordination (having average bond angles of 120xc2x0 and average bond lengths of 1.37 angstroms) to the tetrahedral coordinated anion (having average bond angles of 109xc2x0 and average bond lengths of 1.48 angstroms).
Molecular species having cis or coaxial 1,2-diol and 1,3-diol functionalities, and particularly carbohydrates, are known to complex with immobilized phenylboronate anion, to form cyclic esters under alkaline aqueous conditions (Lorand, J. P. and Edwards, J. O. (1959) J. Org. Chem., 24, 769).
Acidification of 1,2-diol and 1,3-diol complexes to neutral pH is know to release the diol containing species, presumably due to hydrolysis of the cyclic ester. Coplanar aromatic 1,3-diols, like 1,8-dihydroxynaphthalene, are known to complex even under acidic conditions due to the hydrolytic stability of six-membered cyclic boronic acid esters (Sienkiewicz, P. A. and Roberts, D. C. (1980) J. Inorg. Nucl. Chem., 42, 1559-1571). Molecular species having pendant 1,2-hydroxylamine, 1,3-hydroxylamine, 1,2-hydroxyamide, 1,3-hydroxyamide, 1,2-hydroxy-oxime and 1,3-hydroxyoxime functionalities are also known to reversibly complex with phenylboronic acid under alkaline aqueous conditions similar to those associated with the retention of diol containing species (Tanner, D. W. and Bruice, T. C. (1967) J. Amer. Chem. Soc., 89, 6954).
Ortho-substituted acetamidophenylboronic acids have been proposed as potential linkers for selective bioconjugation via the vicinal diol moieties of the carbohydrate residues associated with glycoproteins (Cai, S. X. and Keana, J. F. W. (1991) Bioconjugate Chem., 2, 317-322). Phenylboronic acid bioconjugates derived from 3-isothiocyanatophenylboronic acid have been successfully utilized for appending radioactive technetium dioxime complexes to monoclonal antibodies for use in medical imaging (Linder, K. E., Wen, M. D., Nowotnik, D. P., Malley, M. F., Gougoutas, J. Z., Nunn, A. D. and Eckelman, W. C. (1991) Bioconjugate Chem., 2, 160-170; Linder, K. E., Wen, M. D., Nowotnik, D. P., Ramalingam, K., Sharkey, R. M., Yost, F., Narra, R. K. and Eckelman, W. C. (1991) Bioconjugate Chem., 2, 407-414).
3-Aminophenylboronic acid has been covalently appended to proteins by a variety of chemical methods and the resulting phenylboronic acid bioconjugates tested for their binding of D-sorbitol, D-mannose and glycated hemoglobin (GHb). The interactions proved to be reversible and of very low affinity rendering the bioconjugates of very limited practical use. Similarly, an alkaline phosphatase phenylboronic acid bioconjugate used in an attempted enzyme-linked assay for the detection of GHb failed to detect the presence of glycated protein (Frantzen, F., Grimsrud, K., Heggli, D. and Sundrehagen, E. (1995) Journal of Chromatography B, 670, 37-45).
A novel class of phenylboronic acid reagents and phenylboronic acid complexing reagents have been developed for conjugating biologically active species by exploiting indirect bioconjugation through a reversible boron complex. These reagents and associated conjugates may be used in a manner analogous to Avidin-Biotin and Digoxigenin-anti-Digoxigenin systems. However, unlike the Avidin-Biotin and Digoxigenin-anti-Digoxigenin systems, wherein the viability of the biological macromolecule must be maintained to preserve requisite binding properties, the bioconjugate formed through the boron complex is generally insensitive to significant variations in ionic strength, temperature, the presence of organic solvents, and the presence of chaotropic agents (protein denaturants).
Phenylboronic acid reagents, phenylboronic acid complexing reagents, their conjugates and bioconjugates, as well as methods for their preparation and use are the subject of U.S. Pat. Nos. 5,594,111, 5,623,055, 5,668,258, 5,648,470, 5,594,151, 5,668,257, 5,677,431, 5,688,928, 5,744,627, 5,777,148, 5,831,045, 5,831,046 and 5,837,878.
The present invention relates to a novel class of bifunctional boronic compound complexing reagents useful for the preparation of bioconjugates, and the method of making and using such reagents. In one embodiment, the boron compound is phenylboronic acid, or derivatives thereof, which complex with the complexing reagents of the present invention. In a second embodiment, the boron compound is phenyldiboronic acid, or derivatives thereof, which complex with the complexing reagents of the present invention. Unless otherwise noted, the phrase bifunctional boronic compound complexing reagent is used herein to include the broader class of boron compound complexing reagents which complex with boron compounds, and the phrase phenylboronic acid is used herein to include the broader class of boron compounds which complex with the boron compound complexing reagents, including bifunctional boronic compound complexing reagents. In the present invention, in the place of prior art Avidin-Biotin and Digoxigenin anti-Digoxigenin systems, bifunctional boronic compound complexing reagents comprised of two boron compound complexing moieties can be utilized in conjunction with the boron compound, such as phenylboronic acid reagents (many of which are known in the prior art) to facilitate chemical conjugation and prepare bioconjugates without the use of intermediary biological macromolecules. Bioconjugate preparation often involves the conjugation of several components including, but not limited to, proteins, peptides, polysaccharides, hormones, nucleic acids, liposomes and cells, with each other or with any other molecular species that add useful properties, including, but not limited to, drugs, radionuclides, toxins, haptens, inhibitors, fluorophores, ligands, solid-phase supports, and boron compound complexing reagents conjugates. These various components utilized in bioconjugate preparation will collectively and individually be termed biologically active species or bioactive species.
Two alternative methods for the preparation of bioconjugates derived from bifunctional boronic compound complexing reagents are disclosed below. In the first method, which is comprised of three steps, a reagent having putative boronic compound complexing moieties is first prepared, and then converted into a boronic compound complexing conjugate prior to reaction with a phenylboronic acid conjugate to afford a bioconjugate. In the second method, which is comprised of two steps, a boronic compound complexing conjugate is prepared in a single step, and then reacted with a phenylboronic acid conjugate to afford a bioconjugate.
Reagents suitable for the modification of a bioactive species for the purpose of incorporating a bifunctional boronic compound complexing moiety for subsequent conjugation to a different (or the same) bioactive species having pendant phenylboronic acid moieties are of General Formula I, 
wherein group R is an electrophilic or nucleophilic moiety suitable for reaction of the putative bifunctional boronic compound complexing reagent with a bioactive species, wherein group R2 is selected from one of H and OH moieties, and wherein group R3 is selected from one of an alkyl (e.g., methyl, ethyl, etc.) and a methylene bearing an electronegative substituent.
Group Z is a spacer selected from (CH2)n and CH2O(CH2CH2O)n2, wherein n is an integer of from 1 to 5, and wherein n2 is an integer of from 1 to 4. Each of group Z2 and Z3 is a spacer selected from CH2Ar, CH2CONHCH2Ar, CH2CONH(CH2)n3COxe2x80x94NHCH2Ar, and (CH2)n4NHCO(CH2)n5CONHCH2Ar, wherein the group Ar represents the aromatic ring in the reagent of General Formula I to which the spacer Z2 or Z3 is appended, wherein n3 is an integer of from 1 to 5, wherein n4 is an integer selected from one of 2 and 3, and wherein n5 is an integer of from 1 to 4. It is to be appreciated that, for a given reagent of General Formula I, spacers Z2 and Z3 need not be the same moiety.
Reaction of a reagent of General Formula I with a bioactive species affords a conjugate having pendant putative bifunctional boronic compound complexing moieties (one or more) of General Formula II, 
wherein groups R2, R3, Z, Z2 and Z3 are as were previously defined, and wherein the symbol labeled BAS represents a biologically active species (or bioactive species) that may or may not contain a portion of a reactive moiety (which may itself have a spacer) used to attach the bioactive species to the reagent.
It will be appreciated that, in many embodiments, several identical reagents of General Formula I will react with a single BAS molecule. For example, if the BAS is a protein, many bifunctional boronic compound complexing reagents will react with the protein, each reacting at one of the several sites on the protein which are reactive with the R group.
The conjugate of General Formula II may be further reacted with hydroxylamine (NH2OH) by amidation of the benzoic acid ester moiety to afford a class of bifunctional boronic compound complexing, conjugate, e.g., conjugate with one or more pendant bifunctional boronic compound complexing moieties of General Formula III, 
wherein groups R2, Z, Z2, Z3 and BAS are as were previously defined.
Phenylboronic acid reagents, many of which are known in the prior art, as well as those described in greater detail in my copending application, titled xe2x80x9cPhenyldiboronic Acid Reagents and Complexesxe2x80x9d, filed Aug. 21, 1998, U.S. Ser. No. 09/138,105, which is incorporated herein by reference, may be appended to a biologically active species to afford a conjugate having pendant phenylboronic acid moieties (one or more) of the general formula of General Formula IV, 
wherein group R4 is selected form one of H and B(OH)2 moieties, and wherein the symbol labeled BAS* represents a second bioactive species, that may include a linker portion and that may differ from the bioactive species labeled BAS. The BAS* may also include a portion of a reactive moiety used to attach the bioactive species to the phenylboronic acid reagent.
A conjugate of General Formula III, with at least one biologically active species and having pendent bifunctional boronic compound complexing moieties (one or more), may be complexed with a conjugate of General Formula IV, prepared from a second bioactive species BAS* and having, pendant phenylboronic acid moieties (one or more), to afford a bioconjugate of General Formula V, 
wherein the symbols labeled BAS and BAS*, and groups R2, R4, Z, Z2 and Z3, are as were previously defined. In this manner, biological macromolecules may be conjugated to one another or with other functionalities that impart useful properties.
Alternatively, a second class of reagents suitable for the modification of a bioactive species for the purpose of incorporating a bifunctional boronic compound complexing moiety for subsequent conjugation to a different (or the same) bioactive species having pendant phenylboronic acid moieties are of General Formula VI, 
wherein group R1 is a reactive electrophilic or nucleophilic moiety suitable for reaction of the putative bifunctional boronic compound complexing reagent with a bioactive species, and wherein group R2 is selected from one of H and OH moieties. Group Z is a spacer selected from (CH2)n and CH2O (CH2CH2O)n2, wherein n is an integer of from 1 to 5, and wherein n2 is an integer of from 1 to 4. Each of group Z2 and group Z3 is a spacer selected from CH2Ar, CH2CONHCH2Ar, CH2CONH(CH2)n3CONHCH2Ar, and (CH2)n4NHCO(CH2)n5CONHCH2Ar, wherein the group Ar represents the aromatic ring in the reagent of General Formula VI to which the spacer Z2 or Z3 is appended, wherein n3 is an integer of from 1 to 5, wherein n4 is an integer selected from one of 2 and 3, and wherein n5 is an integer of from 1 to 4. It is to be appreciated that, for a given reagent of General Formula VI, spacers Z2 and Z3 need not be the same moiety.
Reaction of a reagent of General Formula VI with a bioactive species affords a conjugate having pendant boronic compound complexing moieties (one or more) of General Formula VII, 
wherein groups R2, Z, Z2, and Z3 are as were previously defined, and wherein the symbol BAS represents the bioactive species that may or may not contain a portion of a reactive moiety used to attach the bioactive species.
In a manner indistinguishable from that described above for the three-step method, a conjugate of General Formula VII (which is comparable to a conjugate of General Formula IE), may be complexed with a conjugate of General Formula IV, to afford a bioconjugate of General Formula V, 
wherein the symbols labeled BAS and BAS*, and groups R2, R4, Z, Z2 and Z3 are as were previously defined. In this manner, biological macromolecules may be conjugated to one another or with other functionalities that impart useful properties.
Bioconjugates of General Formula V, whether formed, for example, in accordance with either the three-step method or the two-step method described above, may be prepared in buffered aqueous solution or organic solvents. The bioconjugate is formed within a few minutes over a range of temperatures from about 4xc2x0 C. to 70xc2x0 C. The stability of the bioconjugate in aqueous solution at a given pH and temperature is significantly influenced by substituent group R2. Bioconjugates of General Formula V, wherein group R4 is H, are stable in aqueous solutions of approximate pH greater than 3.5 and less than 12.5. Bioconjugates of General Formula V, wherein group R2 is OH, are stable in aqueous solutions of approximate pH greater than 1.5 and less than 12.5.